We have observed that phospholipase A2 (PLA2) activation and arachidonic acid (AA) release are essential for monocyte/macrophage adherence and spreading. In this study, we addressed the relationship between AA release and cell adherence/spreading in murine resident peritoneal macrophages, and the roles of specific PLA2s in these processes. The PLA2-specification inhibitors, (E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one [BEL, specific for the Ca2+ -independent PLA2 (iPLA2)] and methyl arachidonoyl fluorophosphonate [MAFP, specific for the Ca2+-dependent phospholipase (cPLA2) inhibited AA release and cell spreading in a correlated fashion while partially decreasing cell adherence. Cell spreading was normalized by the addition of AA to PLA2-inhibited cells. AA release during adherence/spreading was also inhibited by Ca2+ depletion or protein kinase C (PKC) inhibition, and was accompanied by increased (but transient) phosphorylation of cPLA2. Inhibition of macrophage adherence and spreading, however, only partially inhibited AA release. Moreover, constitutive AA release was seen in fully spread macrophages which was inhibited by BEL, but not MAFP or Ca2+ depletion. BEL also reversed the phenotype of fully spread cells. These data suggest that macrophage spreading requires the release of AA by the IPLA2) which appears to be constitutively active) and cPLA2 (which appears to be stimulated by adherence and spreading). Maintenance of macrophage spreading, in contrast, appears to be principally dependent on the iPLA2